Bacterial Growth assessment by Using Turbidimetric Determination

Aim: To study the different phases of bacterial growth.

To plot standard growth curve of Staphylococcus aureus.
To determine the generation time of given bacteria.
Principle:
Turbidimetric determination is useful for plotting growth curves of bacteria in broth or liquid
media. It is one of the simplest methods used to analyze trends in growth because it uses a
spectrophotometer to track changes in the optical density (OD) over time. In other words: as the
number of cells in a sample increase, the transmission of light through the sample will
decrease. The standard OD setting is 660 nanometers for yellow to brown broth samples, but can
be adjusted if the color is not in this range or if growth is expected to be lower or greater than
average. The following is an example of a standard procedure using this method. It utilizes
spectrophotometric measurements every 30 minutes intervals until the absorbance no longer
increases.

The growth curve has four distinct phases

1. Lag phase
When a microorganism is introduced into the fresh medium, it takes some time to adjust with the
new environment. This phase is termed as Lag phase, in which cellular metabolism is
accelerated, cells are increasing in size, but the bacteria are not able to replicate and therefore no
increase in cell mass. The length of the lag phase depends directly on the previous growth
condition of the organism. When the microorganism growing in a rich medium is inoculated into
nutritionally poor medium, the organism will take more time to adapt with the new environment.
The organism will start synthesising the necessary proteins, co-enzymes and vitamins needed for
their growth and hence there will be a subsequent increase in the lag phase. Similarly when an
organism from a nutritionally poor medium is added to a nutritionally rich medium, the organism
can easily adapt to the environment, it can start the cell division without any delay, and therefore
will have less lag phase it may be absent.
2. Exponential or Logarithmic (log) phase
During this phase, the microorganisms are in a rapidly growing and dividing state. Their
metabolic activity increases and the organism begin the DNA replication by binary fission at a
constant rate. The growth medium is exploited at the maximal rate, the culture reaches the
maximum growth rate and the number of bacteria increases logarithmically (exponentially) and
finally the single cell divide into two, which replicate into four, eight, sixteen, thirty two and so
on (That is 20, 21, 22, 23.........2n, n is the number of generations) This will result in a balanced
growth. The time taken by the bacteria to double in number during a specified time period is
known as the generation time. The generation time tends to vary with different organisms. E.coli
divides in every 20 minutes, hence its generation time is 20 minutes, and for Staphylococcus
aureus it is 30 minutes.

3. Stationary phase
As the bacterial population continues to grow, all the nutrients in the growth medium are used up
by the microorganism for their rapid multiplication. This result in the accumulation of waste
materials, toxic metabolites and inhibitory compounds such as antibiotics in the medium. This
shifts the conditions of the medium such as pH and temperature, thereby creating an
unfavourable environment for the bacterial growth. The reproduction rate will slow down, the
cells undergoing division is equal to the number of cell death, and finally bacterium stops its
division completely. The cell number is not increased and thus the growth rate is stabilised. If a
cell taken from the stationary phase is introduced into a fresh medium, the cell can easily move
on the exponential phase and is able to perform its metabolic activities as usual.

4. Decline or Death phase

The depletion of nutrients and the subsequent accumulation of metabolic waste products and
other toxic materials in the media will facilitates the bacterium to move on to the Death phase.
During this, the bacterium completely loses its ability to reproduce. Individual bacteria begin to
die due to the unfavourable conditions and the death is rapid and at uniform rate. The number of
dead cells exceeds the number of live cells. Some organisms which can resist this condition can
survive in the environment by producing endospores.
Procedure:

1. Prepare sterile broth or media that will be used for inoculation. When ready to use, make
sure the temperature is not too hot. The media container should be comfortable to the
touch.
2. Power on a spectrophotometer and allow it to warm up, preferably for several minutes
before use.
3. Set the wavelength of the spectrophotometer to 660 nm (or another appropriate setting).
Add 5 mL of uninoculated sterile media to a clean cuvette and blank the machine by
setting it to 0 ABS with this sample. This step standardizes the turbidity of the media
without any cells in it, so that further calculations can quantitate growth due to changes in
the turbidity.
4. Inoculate the primary media from which samples will be taken.
5. Immediately after inoculating, take a 5 mL sample of the inoculated media and pipette it
into a clean cuvette. Place it in the blanked spectrophotometer, and record the OD
reading. This reading should be recorded at time “0”.
6. Repeat the previous step at 15 minute intervals until the absorbance no longer increases.
7. Plot the readings on a graph with Time as the X-axis and OD as the Y-axis.

OBDERVATION:

Time in min OD at 660 nm

0 0.005
30 0.04
60 0.20
90 0.40
120 0.61
150 0.80
180 0.92
210 0.94
240 0.95
270 0.95
300 0.95
330 0.95
CALCULATION:

The generation time can be calculated from the growth curve

Calculation of generation time

The exactly doubled points from the absorbance readings were taken and, the points were
extrapolated to meet the respective time axis.

Generation Time = (Time in minutes to obtain the absorbance 0.4) – (Time in minutes to obtain
the absorbance 0.2)

= 90-60

= 30 minutes

Result:
The standard growth curve of Staphylococcus aureus was plotted and the generation time was
found to be 30 min